Restoring Multidrug-Resistant Escherichia coli Sensitivity to Ampicillin in Combination with (-)-Epigallocatechin Gallate

Abstract

Multidrug-resistant (MDR) bacteria, especially Escherichia coli, are a major contributor to healthcare-associated infections globally, posing significant treatment challenges. This study explores the efficacy of (-)-epigallocatechin gallate (EGCG), a natural constituent of green tea, in combination with ampicillin (AMP) to restore the effectiveness of AMP against 40 isolated MDR E. coli strains. Antimicrobial activity assays were conducted to determine the minimum inhibitory concentrations (MIC) of EGCG using the standard microdilution technique. Checkerboard assays were employed to assess the potential synergistic effects of EGCG combined with AMP. The pharmacodynamic effects of the combination were evaluated through time-kill assays. Outer membrane disruption was analyzed by measuring DNA and protein leakage and with assessments using N-phenyl-1-naphthylamine (NPN) and rhodamine 123 (Rh123) fluorescence dyes. Biofilm eradication studies involved biofilm formation assays and preformed biofilm biomass and viability assays. Scanning electron microscopy (SEM) was used to examine changes in cellular morphology. The results indicated that EGCG demonstrated activity against all isolates, with MICs ranging from 0.5 to 2 mg/mL, while AMP exhibited MIC values between 1.25 and 50 mg/mL. Importantly, the EGCG-AMP combination showed enhanced efficacy compared to either treatment alone, as indicated by a fractional inhibitory concentration index between 0.009 and 0.018. The most pronounced synergy was observed in 13 drug-resistant strains, where the MIC for EGCG dropped to 8 µg/mL (from 1 mg/mL alone) and that for AMP to 50 µg/mL (from 50 mg/mL alone), achieving a 125-fold and 1000-fold reduction, respectively. Time-kill assays revealed that the bactericidal effect of the EGCG-AMP combination occurred within 2 h. The mechanism of EGCG action includes the disruption of membrane permeability and biofilm eradication in a dose-dependent manner. SEM confirmed that the combination treatment consistently outperformed the individual treatments. This study underscores the potential of restoring AMP efficacy in combination with EGCG as a promising strategy for treating MDR E. coli infections.

Keywords: EGCG; Escherichia coli; ampicillin; antimicrobial resistant; synergistic.

Figure 1

Effect of EGCG-AMP combination on MDR E. coli E48 strain. Time-kill kinetics for EGCG, ampicillin (AMP), and their combination were analyzed. Bacterial samples were taken at 1, 2, 4, 8, 16, and 24 h to measure viable cell counts. Dashed bars indicate the bactericidal threshold.

Figure 2

Effect of EGCG-AMP combination on membrane permeability. The E. coli E48 strain was exposed to EGCG alone (at 1×, 2×, and 4× MIC), AMP alone, or the EGCG-AMP combination for 1 h at 37 °C. DNA (a) and protein (b) levels were measured. The relative fluorescence intensity (RFI) of NPN (c) and Rh123 (d) was also assessed. Triton X-100 (0.1%) served as the positive control (TX). Significant differences compared to untreated controls are indicated by asterisks (*** p < 0.001).

Figure 3

Effect of EGCG-AMP combination on biofilm formation. The E. coli E48 strain was exposed to EGCG alone (at 1×, 2×, and 4× MIC), AMP alone, or the EGCG-AMP combination for biofilm formation assays, as well as assays measuring preformed biofilm biomass and viability. Biofilm formation (a) and preformed biofilm (b) were evaluated using crystal violet staining. The viability of preformed biofilm (c) was assessed via the MTT assay. Significant differences compared to untreated controls are indicated by asterisks (* p < 0.05, *** p < 0.001).

Figure 4

Effect of EGCG-AMP combination on bacterial cell morphology. The E. coli E48 strain was exposed for 4 h at 37 °C to either EGCG or AMP alone, and to their combination. Scanning electron microscopy (SEM) images at a magnification of 25,000× illustrate (a) the control; (b) EGCG at a concentration of 1 mg/mL; (c) AMP at a concentration of 50 mg/mL; and (d) treatment with the EGCG-AMP combination (EGCG 0.008 mg/mL + AMP 0.05 mg/mL).